Protocol for the separation of extracellular vesicles by ultracentrifugation from in vitro cell culture models.
Protocol for the separation of extracellular vesicles by ultracentrifugation from in vitro cell culture models.
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DOI：
10.1016/j.xpro.2021.100303
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发表时间：
2021-03-19
期刊：
影响因子：
--
通讯作者：
Mercurio AM
中科院分区：
文献类型：
--
作者：
Chhoy P;Brown CW;Amante JJ;Mercurio AM
胞外囊泡(EVS)作为细胞外运输的货物，在运输关键分子成分方面起着关键作用。虽然已经开发了几种从哺乳动物细胞中提取EV的方法，但EV的特定分离方法可以对膜的完整性和产量产生深远的影响。在这里，我们描述了一种使用差速超速离心法将EV从贴壁上皮细胞中分离出来的一步一步的程序。分离的EV可以通过免疫印迹、质谱学和透射电子显微镜进一步分析，以获得EV的产量和形态。有关本议定书的使用和执行的完整细节，请参阅。用超速离心法从细胞培养模型中分离EV EV产量和形态的免疫印迹测定和EV特异性生物标记物的电子显微镜评估以确定EV的浓缩胞外小泡(EV)在运输关键分子成分作为细胞外运输的货物方面起着关键作用。虽然已经开发了几种从哺乳动物细胞中提取EV的方法，但EV的特定分离方法可以对膜的完整性和产量产生深远的影响。在这里，我们描述了一种使用差速超速离心法将EV从贴壁上皮细胞中分离出来的一步一步的程序。分离的EV可以通过免疫印迹、质谱学和透射电子显微镜进一步分析，以获得EV的产量和形态。
Extracellular vesicles (EVs) play key roles in transporting key molecular constituents as cargo for extracellular trafficking. While several approaches have been developed to extract EVs from mammalian cells, the specific method of EV isolation can have a profound effect on membrane integrity and yield. Here, we describe a step-by-step procedure to separate EVs from adherent epithelial cells using differential ultracentrifugation. Separated EVs can be further analyzed by immunoblotting, mass spectrometry, and transmission electron microscopy to derive EV yield and morphology. For complete details on the use and execution of this protocol, please refer to. Description of EV separation from cell culture models using ultracentrifugation Determination of EV yield and morphology by immunoblotting and TEM Assessment of EV-specific biomarkers to determine EV enrichment Extracellular vesicles (EVs) play key roles in transporting key molecular constituents as cargo for extracellular trafficking. While several approaches have been developed to extract EVs from mammalian cells, the specific method of EV isolation can have a profound effect on membrane integrity and yield. Here, we describe a step-by-step procedure to separate EVs from adherent epithelial cells using differential ultracentrifugation. Separated EVs can be further analyzed by immunoblotting, mass spectrometry, and transmission electron microscopy to derive EV yield and morphology.
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影响因子：
16
作者：
Lötvall J;Hill AF;Hochberg F;Buzás EI;Di Vizio D;Gardiner C;Gho YS;Kurochkin IV;Mathivanan S;Quesenberry P;Sahoo S;Tahara H;Wauben MH;Witwer KW;Théry C
通讯作者：
Théry C
DOI：
10.1073/pnas.1521230113
发表时间：
2016-02-23
影响因子：
11.1
作者：
Kowal, Joanna;Arras, Guillaume;Thery, Clotilde
通讯作者：
Thery, Clotilde
影响因子：
4.6
作者：
Patel, Girijesh Kumar;Khan, Mohammad Aslam;Singh, Ajay Pratap
通讯作者：
Singh, Ajay Pratap
影响因子：
4.6
作者：
Wei Z;Batagov AO;Carter DR;Krichevsky AM
通讯作者：
Krichevsky AM
影响因子：
--
作者：
Yu LL;Zhu J;Liu JX;Jiang F;Ni WK;Qu LS;Ni RZ;Lu CH;Xiao MB
通讯作者：
Xiao MB